The present invention is generally in the field of methods for manufacture of recombinant proteins, and especially in the field of refolding of recombinant proteins expressed in the inclusion bodies of procaryotic expression systems such as E. coli. 
Expression of recombinant proteins with natural biological activity and structure, referred to as “proteomics”, becomes increasingly important with the completion of genomic sequencing for several organisms and the near completion of human genome sequencing. One aspect of proteomics is to express large amounts of protein for structural and functional studies, as well as for commercial applications. The least expensive and most efficient way to express recombinant proteins is to express the proteins in E. coli. Proteins are expressed either intracellularly or secreted into the periplasmic spaces. In the former case, the proteins are often deposited in inclusion bodies, especially if the protein has disulfide bonds.
However, one of the problems in expressing mammalian proteins in E. coli is that most of the expressed proteins form insoluble inclusion bodies. While this problem can be circumvented by using various mammalian or insect expression systems, growing E. coli is faster and less expensive compared to mammalian and insect cultures. Moreover, some proteins are toxic to the host when expressed in their native forms, thus expression as insoluble inclusion bodies is the only way to obtain large quantities of recombinant proteins. Importantly, high levels of expression can be achieved for most proteins. 400 to 600 mg of inclusion bodies per liter of bacterial culture can routinely be achieved, with up to 9,700 mg/L having been reported using this method (Jeong K L; Lee S Y, 1999. Appl. Environ. Microbiol 65:3027–32). Inclusion bodies can be easily purified to greater than 90% with a simple freeze/thaw and detergent washing procedure.
Inclusion bodies appear as dense cytoplasmic granules when the cells are observed under a light microscope. Typically, the cells will be lysed by mechanical disruption of the cells, followed by centrifugation for 30 min at 4700 g. Inclusion bodies will sediment at low g forces and can be separated from many other intracellular proteins. Further purification can be done by washing the pellet with the buffer used during the cell disruption, or by centrifuging the resuspended pellet in 40–50% glycerol.
Many extracellular proteins of eukaryotes contain disulfide bonds. Proteins having multiple disulfide bonds may form non-native disulfide bonds during folding from the reduced species. Further folding is then blocked unless the incorrect disulfide bond is cleaved by reduction with an external thiol or by attack from a protein thiol. Eukaryotic organisms that secrete disulfide containing proteins also machinery for ensuring proper disulfide bond formation. A distinct disadvantage of expression of recombinant proteins in prokaryotes as inclusion bodies is that the proteins are not obtained in their native state, and typically are not functionally active. A variety of methods have been used to re-solubilize the proteins and refold them to reform active protein. Dissolution of the pelleted recombinant protein usually requires the use of denaturants such as 7 M guanidine hydrochloride or 8 M urea. The amount of aggregation may continue to increase with time if the protein is allowed to remain in the denaturant (Kelley and Winkler, “Folding of Eukaryotic Proteins Produced in Escherichia coli” Genetic Engineering 12, 1–19 at p. 6 (1990)). Removal of the denaturant from the solubilized inclusion bodies by dialysis or desalting columns will cause the protein to precipitate under conditions where the native protein needs to be refolded. A misfolded protein solution can also have a very low specific activity in biological assays.
Although there are many reports of expression and refolding of various proteins in E. coli as inclusion bodies, one of the misconceptions in protein refolding is that a unique refolding method has to be developed for each individual protein (see Kelley and Winkler at p. 6). Another misconception is that most of the mammalian proteins cannot be refolded from inclusion bodies. (for review, see: Rudolph R., Lilie H., 1996. FASEB J 10:49–56; Lilie H, Schwarz E, Rudolph R. 1998. Curr Opin Biotechnol 9:497–501). Because published works are mostly “success” stories in refolding inclusion bodies from E. coli, it is impossible to get a general idea about what percentage of mammalian proteins can be purified using this procedure.
There are probably more refolding methods than refolded proteins reported in the literature (for review, see: Rudolph R., Lilie, H. 1996, FASEB J 10:49–56; Lilie, H., Schwarz, E., Rudolph, R. 1998, Curr. Opin. Biotechnol. 9:497–501). Different chaperones, detergents, and chaotrophs have been used to help refolding. In addition, pH, ionic strength, temperature, buffer formulation, and reducing/oxidation reagents can all effect refolding. It would be prohibitive to test all these conditions for refolding large amounts of proteins, as required for studies in proteomics or structural genomics.
A single simplified procedure to refold most of the proteins that are expressed in recombinant systems, especially those which form inclusion bodies in systems such as E. coli, is therefore needed.
It is therefore an object of the present invention to provide a “universal” method for refolding of proteins, especially recombinant proteins, especially recombinant proteins present in inclusion bodies in bacterial hosts.